Bottom Line:
Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS).When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins.This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

ABSTRACTHuman tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

f3: Identification of proteins in protein-rich pool by SDS-PAGE in combination with tandem MS (MS/MS). A: aliquots from AS and IS protein-rich pools after density-gradient centrifugation were dialyzed against water, freeze-dried, and solubilized in 8 M urea. Proteins were separated by SDS-PAGE on a 4–20% gradient gel under reducing conditions and visualized by Coomassie blue staining. Positions of molecular mass markers (kDa) are indicated at left. Major bands (1–7 for AS and 1–6 for IS) were digested in-gel with trypsin, and proteins were identified by MS/MS (B). First number in parentheses is number of peptides identified by gel digestion, and second number represents number of peptides identified for the same protein by shotgun analysis. Albumin detected in AS is from a bovine source and is derived from culture medium.

Mentions:
SDS-PAGE analysis of IS and AS protein-rich fractions revealed that the major bands in each sample were not the same (Fig. 3). These major bands (Fig. 3A) were excised from the gel, digested with trypsin, and identified by MS/MS (Fig. 3B). This analysis revealed that polymeric IgG receptor, serum albumin, LPLUNC1, actin, and cytokeratin were major proteins common to both pools and consistent with high peptide coverage (Fig. 3B). In contrast, other major proteins, for example, circulating immunoglobulins and leukocyte elastase, were identified only in the IS sample; however, these two proteins are most likely derived from cell types (B cells and neutrophils) that are not represented in the cell cultures. MS/MS of the tryptic peptides derived from the AS and IS protein-rich fractions identified a total of 130 proteins in AS and 132 in IS (Table 1); 80 proteins are common to AS and IS. Table 1 omits 14 known components of saliva (42, 44) and 24 potential inflammatory cell and serum-derived proteins (according to the Swiss-Prot protein database annotation) found in IS (see the list of all the proteins found in the supplemental Table 1 in the online version of this article).

f3: Identification of proteins in protein-rich pool by SDS-PAGE in combination with tandem MS (MS/MS). A: aliquots from AS and IS protein-rich pools after density-gradient centrifugation were dialyzed against water, freeze-dried, and solubilized in 8 M urea. Proteins were separated by SDS-PAGE on a 4–20% gradient gel under reducing conditions and visualized by Coomassie blue staining. Positions of molecular mass markers (kDa) are indicated at left. Major bands (1–7 for AS and 1–6 for IS) were digested in-gel with trypsin, and proteins were identified by MS/MS (B). First number in parentheses is number of peptides identified by gel digestion, and second number represents number of peptides identified for the same protein by shotgun analysis. Albumin detected in AS is from a bovine source and is derived from culture medium.

Mentions:
SDS-PAGE analysis of IS and AS protein-rich fractions revealed that the major bands in each sample were not the same (Fig. 3). These major bands (Fig. 3A) were excised from the gel, digested with trypsin, and identified by MS/MS (Fig. 3B). This analysis revealed that polymeric IgG receptor, serum albumin, LPLUNC1, actin, and cytokeratin were major proteins common to both pools and consistent with high peptide coverage (Fig. 3B). In contrast, other major proteins, for example, circulating immunoglobulins and leukocyte elastase, were identified only in the IS sample; however, these two proteins are most likely derived from cell types (B cells and neutrophils) that are not represented in the cell cultures. MS/MS of the tryptic peptides derived from the AS and IS protein-rich fractions identified a total of 130 proteins in AS and 132 in IS (Table 1); 80 proteins are common to AS and IS. Table 1 omits 14 known components of saliva (42, 44) and 24 potential inflammatory cell and serum-derived proteins (according to the Swiss-Prot protein database annotation) found in IS (see the list of all the proteins found in the supplemental Table 1 in the online version of this article).

Bottom Line:
Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS).When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins.This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

ABSTRACTHuman tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.